The goal of Project 2 is to understand how TIM proteins regulate immune responses and autoimmunity via their recognition of phosphatidylserine on apoptotic cells. We discovered the Tim (T cell- Imunoglobulin-Mucin) genes, which we showed regulate tolerance, Th2 cytokine production, and asthma. We propose to: Specific Aim 1. Determine the functional consequences of TIM-4 and TIM-3 on APC binding to phosphatidylserine on apoptotic cells. We will examine effects on (a) anti-inflammatory cytokine production: IL-10, TGF-|3; (b) expression of coinhibitory molecules: PD-L1, PD-L2, B7-H3, B7-H4; (c) expression of co-stimulatory molecules: B7-1, B7-2, ICOS-L, CD40; (d) PGE2 and IDO production. Specific Aim 2. Determine the functional consequences of TIM-4 and TIM-3 on APC binding to phosphatidylserine in a pro-inflammatory context and determine the effects of TIM-1 and TIM-3 on T cells binding to PtdSer on an apoptotic APC. We will examine the (a) effects of TIM-4 or TIM-3 mediated phagocytosis of apoptotic versus necrotic cells on APC function in pro-inflammatory conditions; (b) determine the effects on T cell activation and TReg development of TIM-1 and TIM-3 on T cells binding to PtdSer on an apoptotic APC; and (c) determine how the health of the APC regulates T cell responses through TIM-1 and TIM-3. Specific Aim 3. Determine the structure/function relationship of TIM alleles on recognition of phosphatidylserine, phagocytosis, and T cell activation. We will perform a mutational analysis of the TIM-4 phosphatidylserine binding site and functional consequences of these mutations on phagocytosis; determine if HBA and BALB/c alleles of TIM-1 and TIM-3 have different capacities to recognize PtdSer and phagocytose apoptotic cells; and (c) determine how TIM-1 costimulation affects T cell activation in vitro and compare responses of TIM-1-expressing T cells from HBA and BALB/c mice. These studies together will greatly increase our understanding of the function of TIMs in the regulation of tolerance and Th2 responses, and will characterize a crucial regulator of CD4 T cell differentiation and a novel and extremely important asthma susceptibility gene family.